The production of large quantities of proteins for use as therapeutics, additives, and other myriad applications remains a challenge. Large-scale fermentation is a commonly used method, but is expensive and difficult to maintain the required quality and consistency of product. When producing proteins in bacteria, vectors that have a high copy number are generally sought because the amount of protein is often directly proportional to gene dosage.
DNA vaccination, perhaps more precisely called DNA-mediated immunization, refers to the direct introduction into a living species of plasmid or even non-plasmid DNA (or RNA) which is able to cause expression of antigenic protein(s) or peptide(s) in the newly transfected cells. Presentation of DNA into tissues of the host species may be by needle injection, particle bombardment or even orally using various DNA formulations which may be either "naked" DNA, coated microparticles, or via liposomes or biodegradable microcapsules or microspheres. In any case relatively large quantities of purified plasmid DNA are required for production of these vaccines.
Runaway replication plasmid vectors have been developed for expression of genes in bacteria. These vectors are based on plasmids, such as RI in which an antisense RNA transcript is a negative regulator of repA translation and a product of the copB gene, is a repressor of transcription of repA. RepA protein, which functions in cis, is necessary and rate-limiting for replication. The copy number of the plasmid is determined by the relative levels of the antisense RNA and RepA mRNA. If the antisense RNA levels decrease and RepA mRNA increases, expansive plasmid replication results. This is achieved by defined temperature sensitive mutations in the plasmid regulatory region or by insertion of an additional inducible promoter (ex. .lambda. promoter) upstream of repA gene. Plasmid replication is then temperature sensitive and is induced to high levels at 42.degree. C. Moreover, protein synthesis is concomitant with replication, leading to high levels of proteins that are encoded by the vector.
While these runaway-replication plasmid vectors have been used to produce a variety of proteins, including hGCSF and somatotropin, the amount of protein produced has been limited by such factors as the copy number (about 1000 copies per cell), and cell death resulting from runaway replication, thus preventing the use of continuous fermentation techniques. Thus, there is a need for expression vector systems without these limitations.
The present invention discloses genes and gene products from small cryptic plasmids and their use in constructing easily controlled, stable, high copy number replication vectors, and further provides other related advantages.